HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy, and a main challenge in achieving HIV-1 antiretroviral (ART)-free remission is the persistence of these latent viral reservoirs. Quantifying the replication competent viral reservoir is critical to understanding how HIV-1 persists and to measuring how this reservoir changes in response to therapeutic strategies. Quantitative co-culture assay have been developed to measure the replication competent reservoir by activating CD4+ T cells from patients in the presence of feeder cells for viral outgrowth and measurements of HIV-1 proteins or genetic material. However, traditional quantitative co-culture assays are time-consuming, labor-intensive and require parallel reactions in large-well format culture. Novel methods to miniaturize the co- culture platform and to increase sample throughput have the potential significantly to improve the study of HIV latency and could be used to streamline and standardize testing of novel reservoir eradication strategies. We propose to adapt and validate a novel, miniaturized chip-based microfluidic co-culture assay and compare its performance with the traditional co-culture assay. We hypothesize that miniaturizing these assays will allow for higher throughput and be less labor intensive than traditional platforms. The proposed method will also allow for tight control of the growth microenvironment which will help to improve sensitivity and standardize test results between laboratories. Our aims are: (1) adapt a chip-based microfluidic cell culture/expansion protocol for viral co-culture to quantify integrated, replication-competent provirus as infectious units per million cells, and, (2) validate the performance of the novel microfluidic co-culture assay compared with traditional laboratory viral outgrowth assays. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our proposed assay has the potential to be used for a variety of applications involving the isolation and growth of infected human cell subsets and tissues, and we ultimately plan to use the assay to quantify replication competent reservoirs in various cell types and from patients undergoing allogeneic stem cell transplantation and cytoreductive chemotherapy.